The overall goal of this laboratory over the past 20 years has been the elucidation of mechanisms regulating allergic inflammation and thereby identification of novel targets for treating allergic diseases. This proposal will focus on mechanisms regulating human basophil activation and effector function in allergic diseases. Basophils are the least abundant granulocytes in the circulation, and only recently has evidence demonstrated that basophils have potent effector functions that bridge innate and adaptive immunity and contribute to the pathogenesis of both allergic and autoimmune diseases. Preliminary studies demonstrate that IL-3 is a key cytokine for polarizing basophils for effector functions that include degranulation in response to Fc5R1 crosslinking, regulation of cell surface molecule expression, cytokine expression, and immune modulation. Key preliminary findings include: 1) detailed analysis of IL-3 activation of human basophils that demonstrates IL-3-inducible phenotypic subsets of basophils with distinct functional characteristics; 2) the first direct evidence for human basophil expression of the thymic stromal lymphopoietin receptor (TSLPR) and signaling in response to TSLP, which is regulated by IL-3 and IL-33; and 3) evidence for basophil functional tolerance' regulated by IL-3. This proposal will investigate the central hypothesis that targeting IL-3 activation of basophils is an effective strategy for treating basophil-mediated allergic diseases. Studies will use human basophils from healthy controls and patients with allergic diseases, as well as murine basophils from IL-3 deficient and control mice, and state- of-the-art cellular and molecular techniques. The central hypothesis will be investigated by testing the following four corollary hypotheses: Aim 1: Investigate the hypothesis that in the absence of IL-3, basophils are in a state of functional tolerance, and that IL-3 activation of basophils breaks functional tolerance and results in heterogeneous subpopulations which may be either in sequential states of activation or stochastically distinct subpopulations; Aim 2: Investigate the hypothesis that IL-3 activated basophils respond to TSLP and acquire a functional phenotype that is polarized for inducing Th2 immune responses.; Aim 3: Investigate the hypothesis that the IL-3 activation state of basophils determines the sensitivity of basophil activation testing for assessment of clinical atopy versus functional tolerance, and Aim 4: Investigate the hypothesis that antagonism, or lack, of IL-3 will result in basophil functional tolerance in murine models of atopy. These studies have the potential to delineate the mechanisms by which basophils are activated for distinct effector functions, lead to novel assays for clinical evaluation of basophil activation, and provide proof-of-concept for potentially targeting IL-3 as a novel treatment of allergic disorders.